All genomes are potential targets of invasion by molecular parasites such as viruses and transposable elements, and organisms have evolved RNA-directed defense mechanisms to cope with the constant threat of genome invaders (Farazi et al., 2008. Development 135: 1201-1214; Girard and Hannon, 2008. Trends Cell Biol. 18:136-148). The well-known subpathway of RNA silencing referred to as RNAi functions in defense against viruses in eukaryotes (Ding and Voinnet, 2007. Cell 130: 413-426). The RNAi defense response is mediated by short (˜22-nucleotide [nt]) RNAs termed siRNAs. The siRNAs are generated from invading viral RNAs by dsRNA-specific, RNase III-like endonucleases called Dicers (Jaskiewicz and Filipowicz, 2008. Curr. Top. Microbiol. Immunol. 320: 77-97). The mature siRNAs are assembled with host effector proteins and target them to corresponding viral target RNAs to effect viral gene silencing via RNA destruction or other mechanisms (Farazi et al., 2008. Development 135: 1201-1214; Girard and Hannon, 2008. Trends Cell Biol. 18:136-148).
Compelling evidence has recently emerged for the existence of an RNA-mediated genome defense pathway in archaea and numerous bacteria that has been hypothesized to parallel the eukaryotic RNAi pathway (for reviews, see Godde and Bickerton, 2006. J. Mol. Evol. 62: 718-729; Lillestol et al., 2006. Archaea 2: 59-72; Makarova et al., 2006. Biol. Direct 1: 7.; Sorek et al., 2008. Nat. Rev. Microbiol. 6: 181-186). Known as the CRISPR-Cas system or prokaryotic RNAi (pRNAi), the pathway is proposed to arise from two evolutionarily and often physically linked gene loci: the CRISPR (clustered regularly interspaced short palindromic repeats) locus, which encodes RNA components of the system, and the cas (CRISPR-associated) locus, which encodes proteins (Jansen et al., 2002. Mol. Microbiol. 43: 1565-1575; Makarova et al., 2002. Nucleic Acids Res. 30: 482-496; Makarova et al., 2006. Biol. Direct 1: 7; Haft et al., 2005. PLoS Comput. Biol. 1: e60). The individual Cas proteins do not share significant sequence similarity with protein components of the eukaryotic RNAi machinery, but have analogous predicted functions (e.g., RNA binding, nuclease, helicase, etc.) (Makarova et al., 2006. Biol. Direct 1: 7).
Unlike the siRNAs of the eukaryotic RNAi system, the effector RNAs of pRNAi are encoded in the host genome. CRISPR loci encode short (typically ˜30- to 35-nt) invader-derived sequences interspersed between short (typically ˜30- to 35-nt) direct repeat sequences (Bolotin et al., 2005. Microbiology 151: 2551-2561; Mojica et al., 2005. J. Mol. Evol. 60: 174-182; Pourcel et al., 2005. Microbiology 151: 653-663; Godde and Bickerton, 2006. J. Mol. Evol. 62: 718-729; Lillestol et al., 2006. Archaea 2: 59-72; Makarova et al., 2006. Biol. Direct 1:7; Horvath et al., 2008. J. Bacteriol. 190: 1401-1412; Sorek et al., 2008. Nat. Rev. Microbiol. 6: 181-186). Recent studies have provided clear experimental evidence that correlates the presence of virus-specific CRISPR sequences with viral immunity (Barrangou et al., 2007. Science 315: 1709-1712; Brouns et al., 2008. Science 321: 960-964; Deveau et al., 2008. J. Bacteriol. 190: 1390-1400). Furthermore, viral infection has been shown to result in the appearance of new corresponding CRISPR elements in surviving strains (Barrangou et al., 2007. Science 315: 1709-1712; Deveau et al., 2008. J. Bacteriol. 190: 1390-1400). This rapidly adapting CRISPR-based immunity acts within natural microbial populations to promote host cell fitness and to influence microbial ecology (Andersson and Banfield, 2008. Science 320: 1047-1050; Tyson and Banfield, 2008. Microbiol. 10: 200-207).
The primary products of the CRISPR loci appear to be short RNAs that contain the invader targeting sequences, and are termed guide RNAs or prokaryotic silencing RNAs (psiRNAs) based on their hypothesized role in the pathway (Makarova et al., 2006. Biol. Direct 1: 7; Hale et al., 2008. RNA, 14: 2572-2579). RNA analysis indicates that CRISPR locus transcripts are cleaved within the repeat sequences to release ˜60- to 70-nt RNA intermediates that contain individual invader targeting sequences and flanking repeat fragments (FIG. 1A; Tang et al., 2002. Proc. Natl. Acad. Sci. 99: 7536-7541; Tang et al., 2005. Mol. Microbiol. 55: 469-481; Lillestol et al., 2006. Archaea 2: 59-72; Brouns et al., 2008. Science 321: 960-964; Hale et al., 2008. RNA, 14: 2572-2579). In the archaeon Pyrococcus furiosus, these intermediate RNAs are further processed to abundant, stable ˜35- to 45-nt mature psiRNAs (Hale et al., 2008. RNA, 14: 2572-2579).